The primary objective of this proposal is to investigate the biochemical basis for the secretion of hydrocholoric acid by the stomach. The effects of other ions and agents which are known to influence acid secretion would also be investigated at the biochemical level. A K ion-stimulated ATPase associated with a light membrane fraction (buoyant density 1.1-1.13) from the oxyntic glands of gastric mucosa has been strongly implicated in the proton transport process. The molecular mechanism involving the formation of phophorylated intermediates leading to the total ATPase reaction will be studied in detail for a better understanding of this possible transport machinery. To this end, attempts will be made to purify this enzyme. The light membrane fraction will be separated into the "right-side-out" and "inside-out" vesicles using the available techniques, such as affinity chromatography and free-flow electrophoresis. Vectorial orientation of the K ion-ATPase in these isolated membrane vesicles with respect to the K ion-effector site and catalytic site will be carefully studied. The relationship between the intrinsic cyclic AMP regulated protein kinase and the transport of different ions in the vesicles will be explored. The subcellular fractionations of the mucosal homogenate would involve differential centrifugation and equilibrium sucrose density gradient centrifugation. The various membrane fractions from the gradient would be characterized by the distribution of K ion-ATPase, NA ion-K ion-ATPase, adenylate cyclase, K ion-PNPPase, 5'-nucleotidase, cytochrome c oxidase and succinic dehydrogenase. Chemical characterization would include phospholipid to cholesterol molar ratio, RNA and DNA content, S.D.S. polyacrylamide gel electrophoresis, high voltage electrophoretic separation and characterization of the peptic and tryptic fragments of the 32P-intermediate, etc. The 32P-intermediate formed after incubation of the light membrane fraction with (gamma-32P) ATP will be separated from other radioactive materials by thorough washing with 5 percent PCA containing 10 mM Pi and ATP on filter paper discs (Whatman #3).